The glutathione S-transferases (GSTs) are a family of detoxification enzymes that metabolize environmental xenobiotics and drugs, including anti-cancer agents, by conjugating them to the tripeptide glutathione (GSH). GSTs also modulate oxidative stress by metabolizing lipid hydroperoxides and lipid hydroxy-enals. GSTs are likely to play a role in sensitivity to atherosclerosis, cataracts, and neurodegenerative diseases. As a canonical family of structurally related proteins, the GSTs provide a model for understanding the evolution of substrate diversity, which apparently correlates with the evolution of protein dynamics in some GSTs. The GSTA1-1 isoform has two unusual features that may uniquely contribute to its catalytic diversity as a detoxification enzyme. One feature is a catalytic Tyr with an unusually low pKa, which, possibly, provides electrostatic forces and increases solvation of the active site. The ionization state of this Tyr does not change during chemical steps of the catalytic cycle, and the function of the unusual ionization properties remains unknown. The second feature is a dynamic C-terminal helix, which undergoes ligand-dependent redistribution between 'open' and 'closed' conformations. Highly related, nearly structurally identical, GSTs possess C-terminal helices that are 'static' and remain either 'open' or 'closed.' This proposal explores the catalytic function of the Tyr ionization properties and of the C-terminal helix and, in particular, the hypothesis that the two features have co-evolved as an evolutionary bridge between primitive GSTs and highly evolved substrate specific isoforms. In order to understand the structure, function, and dynamics of the GST family, the specific aims of this proposal are: 1) to determine the stage of GSTA1-1 catalysis at which the C-terminus closes; 2) to determine the function of the unusual ionization properties of the active site Tyr; 3) to explore the molecular determinants of substrate diversity by directed evolution of GSTA1-1 and directed de-evolution of GSTA4-4. The techniques to be used include x-ray crystallography, NMR, and fluorescence of model ternary complexes, to monitor the C-terminal structure and dynamics. In order to determine whether the C-terminus must be closed in the transition state for the chemical step, linear free energy relationships will be exploited. The relationship, if any, between C-terminal dynamics and the ionization of the catalytic Tyr will be explored with stopped-flow kinetic approaches and steady state fluorescence with an engineered Trp reporter.